3&#39;-[(2Z)-[1-(3,4-DIMETHYLPHENYL)-1,5-DIHYDRO-3-METHYL-5-OXO-4H-PYRAZOL-4-YLIDENE]HYDRAZINO]-2&#39;-HYDROXY-[1,1&#39;-BIPHENYL]-3-CARBOXYLIC ACID bis-(MONOETHANOLAMINE)

ABSTRACT

An improved thrombopoietin mimetic, the bis-(monoethanolamine) salt of 3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylic acid.

This application is a continuation of U.S. application Ser. No.14/050,976 filed Oct. 10, 2013, which is a continuation of U.S.application Ser. No. 13/334,770 filed Dec. 22, 2011, which is acontinuation of U.S. application Ser. No. 12/873,565, filed Sep. 1,2010, now U.S. Pat. No. 8,088,813, which is a continuation of U.S.application Ser. No. 12/472,819, filed May 27, 2009, now U.S. Pat. No.7,795,293, which is a continuation of U.S. application Ser. No.10/515,304, filed Feb. 22, 2006, now U.S. Pat. No. 7,547,719, which is a371 of International Application No. PCT/US03/016255, filed May 21,2003, which claims the benefit of U.S. Provisional Application No.60/382,871, filed May 22, 2002.

This invention relates to an improved thrombopoietin (hereinafter TPO)mimetic, the bis-(monoethanolamine) salt of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid. The compound is represented by Structure I:

The compound of this invention is useful as an agonist of the TPOreceptor, particularly in enhancing platelet production.

DETAILED DESCRIPTION OF THE INVENTION

3′-{N′-[1-(3,4-dimethylphenyl)-3-methyl-5-oxo-1,5-dihydropyrazol-4-ylidene]hydrazino}-2′-hydroxybiphenyl-3-carboxylicacid is a compound which is disclosed and claimed, along withpharmaceutically acceptable salts, hydrates, solvates and estersthereof, as being useful as an agonist of the TPO receptor, particularlyin enhancing platelet production and particularly in the treatment ofthrombocytopenia, in International Application No. PCT/US01/16863,having an International filing date of May 24, 2001; InternationalPublication Number WO 01/89457 and an International Publication date ofNov. 29, 2001, the entire disclosure of which is hereby incorporated byreference.

It has now surprisingly been found that the bis-(monoethanolamine) saltof3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid has numerous advantages over the free acid. The free acid is poorlysoluble in water (approximately 5 micrograms per milliliter). This poorsolubility adversely affects the ability of the free acid to beformulated into pharmaceutical dosage forms and reduces thebioavailability of the compound in vivo.

While the free acid is highly useful as an agonist of the TPO receptor,particularly in enhancing platelet production and particularly in thetreatment of thrombocytopenia, the bis-(monoethanolamine) salt of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid has the added advantages of enhanced solubility andbioavailability.

The compound of this invention,3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine) (hereinafter—“Active Ingredient”), is usefulas an agonist of the TPO receptor, particularly in enhancing plateletproduction and particularly in the treatment of thrombocytopenia. TheActive Ingredient can be administered in a conventional dosage formprepared by combining the Active Ingredient with a conventionalpharmaceutically acceptable carrier or diluent according to techniquesreadily known to those of skill in the art, such as those described inInternational Application No. PCT/US01/16863. The route ofadministration may be oral, parenteral or topical. The term parenteralas used herein includes intravenous, intramuscular, subcutaneous,intranasal, intrarectal, intravaginal or intraperitoneal administration.Oral administration is generally preferred.

As used herein the term “monoethanolamine” means “2-aminoethanol”. Dosesof the presently invented Active Ingredient in a pharmaceutical dosageunit as described above will be an efficacious, nontoxic quantitypreferably selected from the range of 0.001-100 mg/kg of total bodyweight, preferably 0.001-50 mg/kg. When treating a human patient in needof a TPO mimetic, the selected dose is administered preferably from 1-6times daily, orally or parenterally. Preferred forms of parenteraladministration include topically, rectally, transdermally, by injectionand continuously by infusion. Oral dosage units for human administrationpreferably contain from 0.05 to 3500 mg of Active Ingredient, mostpreferably from 0.5 to 1,000 mg of Active Ingredient. Oraladministration, which uses lower dosages is preferred. Parenteraladministration, at high dosages, however, also can be used when safe andconvenient for the patient. The above dosages relate to the preferredamount of the Active Ingredient expressed as the free acid.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of the Active Ingredient willbe determined by the nature and extent of the condition being treated,the form, route and site of administration, and the particular patientbeing treated, and that such optimums can be determined by conventionaltechniques. It will also be appreciated by one of skill in the art thatthe optimal course of treatment, i.e., the number of doses of the ActiveIngredient given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

Generally speaking, the compound of this invention is prepared bydissolving the free acid,3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid, in an appropriate organic solvent, preferably tetrahydrofuran(hereinafter THF) or ethanol/IMS (Industrial Methylated Spirit),filtering the resultant mixture to remove contaminants, then adding thissolution to a solution of two or more equivalents of ethanolamine in anorganic solvent, preferably a water-miscible solvent, which may containa measured amount of water, preferably up to 5 volumes of water withrespect to the free acid. The compound of this invention is filtered offand dried, for example, dried in vacuo or air dried at an elevatedtemperature.

Ethanolamine, 99%, was purchased from the Aldrich Chemical Company,Milwaukee, Wis.

Tetrahydrofuran (THF) and Industrial Methylated Spirit 74 O.P. (IMS)were purchased from BDH Laboratory Supplies, Poole, England.

The following examples further illustrate the present invention. Theexamples are not intended to limit the scope of the invention as definedhereinabove and as claimed below.

EXAMPLE 1 Preparation of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid-carboxylic acid bis-(monoethanolamine)

3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid, 1 g of crude orange solid, in 16.75 ml of THF was stirred atapproximately 30° C. Water (2.0 ml) was added slowly to maintain atemperature greater than 28° C. When the addition was complete, thetemperature was returned to 30° C. and the solution filtered through aglass fibre pad (2×Whatman GFC filters) to remove particulate matter.The filter was washed through with THF (2.0 ml) which was added to thefiltrate. The filtrate was allowed to cool to room temperature.Ethanolamine (0.324 g, 2.35 mol. equiv.) was dissolved in IMS (26 ml)and stirred under a nitrogen atmosphere at room temperature. Thefiltrate containing the free acid was added to the ethanolamine solutionover 20 to 30 minutes. The resulting dark red suspension was stirred for3 hours and the solid isolated by filtration and dried at 50° C. in avacuum oven over night to yield 1.22 g (96%) of the title compound.

Proton NMR (400 MHz, DMSO-d6+20 ul TFA, referenced to DMSO-d5 δ2.5):δ2.21 (s, 3H), 2.26 (s, 3H), 2.31 (s, 3H), 2.85 (m, 4H), 3.57 (t, 4H),7.07 (m), 7.14 (s), 7.18 (d, overlapped 3H), 7.61 (t), 7.63 (dd,overlapped 2H), ˜7.7 (m, overlapped 2H), 7.79 (d), ˜7.8 (br. s,overlapped 2H), 7.96 (d, 2H), 8.13 (s, 1H), 13.8 (br. s, not measurable,superimposed on TFA resonance) and signals for THF 1.76 (m) and 3.60(overlayed by ethanolamine signal) integrating at 1.05% w/w and forethanol 1.06 (t) and 3.44 (q) integrating at 1.3% w/w.

IR Data (Nujol mull)1636, 1506, 1466, 1378, 1348, 1294, 1273, 1255, 1228, 1194, 1127, 1118,1066, 1015, 767, 747 cm⁻¹.

EXAMPLE 2 Preparation of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine)

3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid, 8 g of crude orange solid, was dissolved at room temperature inTHF (240 ml) in a 500 ml round bottom 3-necked flask under a nitrogenatmosphere. Ethanolamine (2.2 ml , 2 molar equivalents) was added viasyringe over 5 minutes. The resulting dark red suspension was stirred atroom temperature for 1.5 hours and the solid isolated by filtration,washed with THF (16 ml×2) and dried at 50° C. in a vacuum oven overnight to yield 10.37 g of the title compound (more than quantitativeyield due to residual solvent—approximately 2.4% w/w THF as determinedby NMR, otherwise similar to Example 1).

EXAMPLE 3 Preparation of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine)

3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid, 8 g of crude orange solid, was suspended at room temperature inethanol (800 ml) under a nitrogen atmosphere. Ethanolamine (2.2 ml, −2molar equivalents) was added via syringe over 5 minutes. The resultingdark red suspension was stirred at room temperature for 45 hours and thesolid isolated by filtration, washed with ethanol (10 ml×2) and dried at50° C. in a vacuum oven over night to yield 9.83 g (96% yield) of thetitle compound. NMR similar to Example 1; ethanol content 1.3% w/w butno THF present.

EXAMPLE 4 Preparation of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine)

3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid (259.0 g) was stirred in THF (4660 ml) at room temperature untilcompletely dissolved. The solution was filtered and the reactor washedwith more THF (520 ml) via the filter. (Combined filtrate=Solution 1).

Meanwhile, another reactor was set up for atmospheric distillation withoverhead stirring. The reactor was charged in turn with IMS 74 O.P.(7770 ml) and then ethanolamine (354 ml). The solution was stirredvigorously and heated until the contents started to distil (BP. 76-77°C.).

Solution 1 was transferred to the dropping funnel fitted to thisreactor. When the contents of the reactor were distilling at a constantrate (ca. 50 ml distillate collected), Solution 1 was added from thedropping funnel at about the same rate or slightly slower than thedistillation rate. On completion of the addition the dropping funnel waswashed through with IMS (260ml×2) ensuring that all the free acid waswashed into the reaction mixture. The apparatus was rearranged forreflux and the resulting dark red suspension stirred at reflux undernitrogen for 30 minutes. It was allowed to cool slowly (overnight) toroom temperature (ca. 20° C.) with stirring under nitrogen.

The suspension was filtered and the dark purple solid washed on thefilter with IMS (520 ml×2). It was vacuum dried at room temperature,then dried at 50° C. in a vacuum oven over night. Weight yield=323.9 g,98%. Residual solvents (GCS) THF=<0.05%, ethanol=0.12%.

The title compound displayed NMR and IR spectra essentially as indicatedin Example 1 with only traces of solvent present.

EXAMPLE 5 Relative Solubilities

The solubility of3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid as the free acid (Compound A) and as the bis-(monoethanolamine)salt (Compound B) was determined in three different systems: water, 0.1HCl and methanol. The data are summarized in Table 1 below.

TABLE 1 Solvent Compound A mg/ml Compound B mg/ml Solubility at 25 degmg/ml mg/ml Water <0.001 14.2 0.1% HCl <0.001 <0.001 methanol 1.9 6.4

The present invention includes within its scope pharmaceuticalcompositions comprising3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine), as the active ingredient, in associationwith a pharmaceutically acceptable carrier or diluent. The compound ofthis invention can be administered by oral or parenteral routes ofadministration and can be formulated in dosage forms appropriate foreach route of administration including capsules, tablets, pills, powdersand granules. In such solid dosage forms, the active compound is admixedwith at least one inert diluent. The oral dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating agents, glidants and antioxidants. In thecase of capsules, tablets and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared for asustained release.

Preparations according to this invention for parenteral administrationinclude sterile aqueous solutions although nonaqueous suspensions ofemulsions can be employed. Such dosage forms may also contain adjuvantssuch as preserving, wetting, osmotic, buffering, emulsifying anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria retaining filter, by incorporating sterilizing agentsinto the compositions, irradiating the compositions or by heating thecompositions.

The following examples further illustrate the pharmaceuticalcompositions which are a feature of this invention.

EXAMPLE 6 Tablet Composition

Lactose, microcrystalline cellulose, sodium starch glycolate, magnesiumstearate and3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine) are blended in the proportions shown inTable 2 below. The blend is then compressed into tablets.

TABLE 2 INGREDIENT mg. 3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5- 8.45dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′- biphenyl]-3-carboxylic acid bis-(monoethanolamine) microcrystalline cellulose 112 lactose 70 sodiumstarch glycolate 8 magnesium stearate 2

EXAMPLE 7 Injectable Parenteral Composition

An injectable form for administering3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine) is produced by stirring 5.0 mg. of thecompound in 1.0 ml. of normal saline.

While the preferred embodiments of the invention are illustrated by theabove, it is to be understood that the invention is not limited to theprecise instructions herein disclosed and that the right to allmodifications coming within the scope of the following claims isreserved.

What is claimed is:
 1. The compound3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine).
 2. A pharmaceutical composition comprising3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid bis-(monoethanolamine) and a pharmaceutically acceptable carrier ordiluent.
 3. A method of treating thrombocytopenia in a mammal, includinga human, in need thereof which comprises administering to such mammal atherapeutically effective amount of a compound as described in claim 1.4. A method as claimed in claim 3, wherein the mammal is a human.
 5. Amethod of enhancing platelet production in a mammal, including a human,in need thereof which comprises administering to such mammal atherapeutically effective amount of a compound as described in claim 1.6. A method as claimed in claim 5, wherein the mammal is a human.
 7. Amethod of agonizing the TPO receptor in a subject which comprisesadministering an effective amount of a compound as described in claim 1.8. A process for preparing a pharmaceutical composition containing apharmaceutically acceptable carrier or diluent and an effective amountof a compound as described in claim 1, which process comprises bringingthe compound described in claim 1 into association with thepharmaceutically acceptable carrier or diluent.
 9. The method of claim 3further comprising co-administering a therapeutically effective amountof an agent selected from the group consisting of: a colony stimulatingfactor, cytokine, chemokine, interleukin or cytokine receptor agonist orantagonists, soluble receptors, receptor agonists or antagonistantibodies, or small molecules or peptides that act by the samemechanisms of one or more of said agents.
 10. A method for enhancingplatelet production obtained from a donor which comprises administeringto such donor a therapeutically effective amount of a compound asdescribed in claim 1 prior to platelet pheresis, blood donation orplatelet donation.
 11. A method for enhancing the number of peripheralblood stem cells obtained from a donor which comprises administering tosuch donor a therapeutically effective amount of a compound as describedin claim 1 prior to leukapheresis.
 12. An in vitro or ex vivo method forenhancing stimulation of megakaryocyte maturation and/or plateletproduction which comprises adding an effective amount of a compound asdescribed in claim 1 to the culture medium of cells that express the TPOreceptor.
 13. An in vitro or ex vivo method for enhancing stimulation ofmegakaryocyte maturation and/or platelet production which comprisesadding an effective amount of a compound as described in claim 1 to theculture medium of stem cells, bone marrow cells, cord-blood cells orperipheral blood cells.
 14. A method of claim 13, wherein themegakaryocytes or platelets are returned to the mammal followingchemotherapy or radiation therapy.
 15. An in vitro or ex vivo method forenhancing the survival and/or proliferation of stem cells, bone marrowcells, cord-blood cells, peripheral blood cells or other types of cellsexpressing the TPO receptor in culture which comprises culturing saidcell in a medium containing an effective amount of a compound asdescribed in claim
 1. 16. A method of claim 15 wherein the stem cellsare returned to the mammal following chemotherapy or radiation therapy.17. A method of treating of neutropenia in a mammal, including a human,in need thereof which comprises administering to such mammal atherapeutically effective amount of a compound as described in claim 1.18. An in vitro or ex vivo method for enhancing stimulation ofneutrophil production which comprises adding an effective amount of acompound as described in claim 1 to the culture medium of stem cells,bone marrow cells, cord-blood cells, peripheral blood cells or othertypes of cells expressing the TPO receptor.
 19. A method of claim 18,wherein the neutrophils are returned to the mammal followingchemotherapy or radiation therapy.
 20. A process for preparing thecompound of claim 1, which process comprises: i) dissolving 3′-[(2Z)-[1-(3,4-dimethylphenyl)-1,5-dihydro-3-methyl-5-oxo-4H-pyrazol-4-ylidene]hydrazino]-2′-hydroxy-[1,1′-biphenyl]-3-carboxylicacid in an appropriate organic solvent, to form a solution; ii) addingtwo or more equivalents of ethanolamine to the solution; and iii)isolating the prepared compound.